226 JOURNAL OF COSMETIC SCIENCE 6-phenolic hydrogen to the oxygen radicals. The free OH group functions as a scavenger of free radicals or singlet oxygen, usually being oxidized itself in the process. The hydroxyl group can be protected from oxidation by esterification with the carboxyl group of an organic acid, usually acetic acid, forming esters such as acetate or succinate derivatives, which are devoid of antioxidant activity. tx-Tocopheryl acetate, a liquid oil, when applied to the skin, acts as a prodrug and has to release the active tx-T by an enzyme-catalyzed hydrolytic reaction. The skin is a viable membrane that can metabolize topically applied substances before they reach the systemic circulation. The metabolizing potential of skin for actives has been estimated to be about 2% of that of liver (1). Cutaneous metabolism can signifi- cantly influence delivery through skin. Trevithick and Mitton (2) showed that about 4.5-5% of tx-TAc was hydrolyzed to free tx-T in mouse skin within 24 hours. Norkus et al. (3) reported that following repeated application of ot-TAc to mouse skin, the concentration of free tx-T in the skin was elevated. We found tx-TAc to be metabolized into free tx-T in viable pig skin, although no metabolism was detected in the stratum corneum (4). However, Alberts et al. (5) have documented substantial skin (epidermal and dermal) absorption of tx-TAc without evidence of bioconversion within the skin to its unesterified form. Micro-Yucatan pig skin was chosen as the animal model for these in vitro studies. Fundamental knowledge of skin metabolism has been obtained by work on the kinetics of epidermal proliferation, and Meyer and Neurand (6) have pointed out similarities between the domestic pig and human epidermal tissues. The authors also found non-specific esterases to be located in the epidermis, subcutis, hair follicles, dermis, and the sebaceous and apocrine glands of pig skin. The formulation vehicle may modify properties of the stratum corneum (e.g., increased hydration), which could influence the penetration of active ingredients (7). The effect of the delivery system on the permeation and metabolism of tx-TAc and tx-T has not been systematically characterized thus far. The extent and speed with which a pharmacologi- cally active substance penetrates the skin depends on the effect that the three compo- nents (vehicle, skin, and active) exert on the diffusion process. The active incorporated in the vehicle should reach the surface of the skin at a suitable rate and concentration. If the site of action lies in the deeper layers of the epidermis or below, the substance must cross the stratum corneum. In addition, it is imperative that a dermal prodrug be metabolized in the skin to release the active molecule. This study was done to (i) formulate tx-TAc in various topical delivery systems and (ii) find the effect of formu- lation factors on the permeation and metabolism of tx-TAc. MATERIALS AND METHODS CHEMICALS AND INSTRUMENTS D-tx-tocopheryl acetate, Covitol © 1360, was obtained as a gift from Henkel Nutrition and Health Group (Illinois). D-tx-tocopherol was obtained as a gift from Archer Daniels Midland Company (Illinois). The following chemicals were obtained directly from the manufacturer and used without purification: SD alcohol (Eastman, Tennessee) isopropyl myristate and mineral oil (Sigma Chemical Company, New Jersey) diisopropyl adipate and isocetyl alcohol, Ceraphyl © 230 and Ceraphyl ©, respectively (ISP Vandyk, New

DELIVERY AND METABOLISM OF ot-TAc 227 Jersey) carbomer Carbopol ©, (BF Goodrich, Ohio) Bis (2-hydroxyethyl)-ammonium hexadecyl hydrogen phosphate, DEA-cetyl phosphate, Amphisol © (Roche Vitamins and Fine Chemicals, New Jersey) diazolidinyl urea, Germall © (Sutton Laboratories, New Jersey) Ethomeen C/25 (Akzonobel, Illinois) and hydroxypropyl cellulose, Klucel © (Hercules Co., Germany). Tween © 20, polyoxyethylene (20) sorbitan monolaurate, and Tween © 80, polyoxyethylene (20) sorbitan monooleate were from ICI surfactants (Dela- ware). Transcutol, ethoxy diglycol, was obtained from Gattefoose (France). Special 30-ml centrifuge tubes were obtained from Corning Inc. The solvents acetonitrile, chloroform, and n-hexane of HPLC grade were obtained from Fisher Scientific (Springfield, NJ). Water refers to freshly deionized water. The centrifuge used was Fisher Centrific. Or- ganic solvents were evaporated using a Savant Speed Vac ©, SC 110. The wrist-action shaker used was from Burrell (Ohio). Vortexing was done with a Vortex-Genie (New York). Particle size analysis was carried out with a Bookhaven Instruments Corporation particle size analyzer where 20 ul of the microemulsion was diluted with 480 lal of water before size determination. The mechanical shaker bath used was a Dubnoff metabolic shaking incubator (Precision Scientific, Illinois). TOPICAL FORMULATIONS All formulations, listed in Tables I and II, were prepared on a weight/weight basis. A simple isopropyl myristate (IPM) solution of tx-TAc (5%) was also prepared. The sta- bility of formulations was assessed by HPLC immediately after preparation and after storage for three weeks at room temperature. Gels were prepared by shaking with the gelling agent for eight hours on a shaker bath at room temperature. The aqueous phase and oily phases of emulsions were heated to 55 øC, after which the former was added to the latter and agitated to produce creamy emulsions. Clear microemulsions were made using a simple vortexing procedure. RECEPTOR FLUID In the metabolism study the receptor media must serve two important functions, viz., maintaining the viability of skin tissue and ensuring adequate solubility of the prodrug and its metabolite. Dulbecco's modified phosphate-buffered saline (DMPBS) was used to maintain tissue viability (8,9). As compounds that are essentially insoluble in water (like the tx-T derivatives) may not partition freely from excised skin into an aqueous receptor fluid, bovine serum albumin (3%) was added to DMPBS (8,10). Fresh deionized water Table I Gel Formulations Used in This Study Gel 1 Gel 2 Gel 3 Ingredients (% w/w) (% w/w) (% w/w) c•-TAc 5 5 5 SD alcohol 92 87 67 Isocetyl alcohol -- -- 12 PEG-15 cocamine 5 5 Hydroxypropyl cellulose 3 3 3 Water -- 8